Selective ablation of diploid embryos

ABSTRACT

Methods of selecting haploid embryos are disclosed. Methods of producing haploid embryos and non-viable diploid embryos on a plant are provided. Methods for selecting haploid embryos produced from haploid inducer maize lines are provided. Methods for producing improved maize haploid inducer lines are disclosed. Maize haploid inducer lines comprising transgenes causing ablated or abnormal diploid embryos are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and hereby incorporates byreference, provisional application 60/887,828 filed Feb. 2, 2007.

THE INVENTION

The present invention relates to the field of plant breeding and plantbiotechnology.

BACKGROUND OF THE INVENTION

Homozygous plants are basic for product development andcommercialization of plants. To obtain homozygous plants requiresseveral generations of self-pollination and segregation analysis. Thisis an inefficient use of labor and time. It would therefore be useful todevelop a method to reduce hand pollination steps normally required toobtain a homozygous plant and reduce the amount of time required toobtain a homozygous population of plants. One way to obtain homozygousplants without the need to self-pollinate multiple generations is toproduce haploids and then double the chromosomes to form doubledhaploids. A process to assist in the selection of haploid embryos andelimination of diploid embryos would increase the efficiency of doubledhaploid production.

SUMMARY OF THE INVENTION

Methods for identifying haploids by preventing the growth of diploidembryos are provided. Methods for identifying haploid plants, seeds,embryos, and plant cells are provided. Methods for producing haploidinducer lines and the haploid inducer lines are provided.

DETAILED DESCRIPTION OF THE INVENTION

A haploid plant has a single set (genome) of chromosomes and the reducednumber of chromosomes (n) in the haploid plant is equal to that in thegamete.

A diploid plant has two sets (genomes) of chromosomes and the chromosomenumber (2n) is equal to that in the zygote.

A haploid cell is one with a single genome, male or female.

A doubled haploid or doubled haploid plant or cell is one that isdeveloped by the doubling of a haploid set of chromosomes. A plant orseed that is obtained from a doubled haploid plant that is selfed anynumber of generations may still be identified as a doubled haploidplant. A doubled haploid plant is considered a homozygous plant. A plantis considered to be doubled haploid if it is fertile, even if the entirevegetative part of the plant does not consist of the cells with thedoubled set of chromosomes. For example, a plant will be considered adoubled haploid plant if it contains viable gametes, even if it ischimeric.

A “haploid embryo” is defined as the embryo formed after one spermnucleus from a pollen grain fuses with the polar nuclei in the embryosac to create a triploid (3N) endosperm and the embryo forms without thecontribution of the male genome.

An “immature haploid embryo” is defined as the embryo formed after onesperm nucleus from a pollen grain fuses with the polar nuclei in theembryo sac to create a triploid (3N) endosperm and before dry down. Andthe embryo forms without the contribution of the male genome.

A “doubled haploid embryo” is an embryo that has one or more cells thatcontain 2 sets of homozygous chromosomes.

“Callus” refers to a dedifferentiated proliferating mass of cells ortissue.

The phrases “contacting”, “comes in contact with” or “placed in contactwith” can be used to mean “direct contact” or “indirect contact”. Forexample, the medium comprising a doubling agent may have direct contactwith the haploid cell or the medium comprising the doubling agent may beseparated from the haploid cell by filter paper, plant tissues, or othercells thus the doubling agent is transferred through the filter paper orcells to the haploid cell.

The term “medium” includes compounds in liquid, gas, or solid state.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. “Plant cell”, as used herein includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores.

The haploid inducer line comprises; 1) a pollen lethality polynucleotideor a non-transmission pollen polynucleotide, 2) an embryo lethalitypolynucleotide, and 3) an embryo lethality repressor.

The pollen lethality polynucleotide or a non-transmission pollenpolynucleotide indicates any polynucleotide that prevents the pollenfrom achieving fertilization. The pollen non-transmission is mosteffective if it occurs at the post-meiotic or gametophytic stage. Thenon-transmission pollen polynucleotide can work through variousmechanisms. It may prevent the pollen from being viable for example bythe expression of a toxic compound.

The embryo lethality polynucleotide is any polynucleotide that preventsproper development of the embryo or causes the embryo to be non-viable.The embryo lethality polynucleotide may slow the growth of the embryo sothat one may be able to distinguish an embryo that carries the embryolethality polynucleotide from an embryo that does not carry the embryolethality polynucleotide.

The embryo lethality repressor is any polynucleotide that when expressedcauses the embryo lethality polynucleotide to be non-effective andallows a viable embryo to develop. This can be achieved through variousmechanisms. For example the embryo lethality repressor can detoxify themolecule expressed by the embryo lethality polynucleotide. An embryorepressor protein may be expressed that inhibits the lethality ofanother protein for example through a protein-protein interaction.Another way the embryo repressor may work is to deter or prevent theexpression of the embryo lethality polynucleotide, for example byexpressing a molecule that binds to the promoter of the lethalitypolynucleotide and blocks some or all expression. Another example thatcould be used in the system is gene silencing. For example, the embryorepressor may be a polynucleotide that prevents the embryo lethalpolynucleotide from functioning through gene silencing.

The embryo lethality polynucleotide and the embryo lethality repressorpolynucleotide are not limited to being expressed only in the embryo.The genes can be expressed in other tissues. It is more effective if theembryo lethality polynucleotide is not expressed in the endosperm sothat the haploid seed can develop normally. Another consideration forthe method to work at its optimum is that the embryo lethalitypolynucleotide expression should to be matched by the expression of theembryo lethality repressor polynucleotide. Anytime the polynucleotidesare inherited together the embryo lethality repressor polynucleotideshould be expressed at a level to deter the negative effects of theembryo lethality polynucleotide.

Typically, the non-transmission pollen polynucleotide and the embryolethality repressor are linked in the developed inducer line. They maybe tightly linked. For the most convenience the non-transmission pollenpolynucleotide and the embryo lethality repressor should be adjacent toeach other, for example on the same construct. For efficient results thenon-transmission pollen polynucleotide and the embryo lethalityrepressor will segregate together. Also typically the non-transmissionpollen polynucleotide and the lethality repressor are linked, but are ata different location than the embryo lethality polynucleotide. Forefficient results, the embryo lethality polynucleotide will segregatefrom the non-transmission pollen polynucleotide and the embryo lethalityrepressor. For the greatest efficiency, the embryo lethalitypolynucleotide is not linked to the non-transmission pollenpolynucleotide and the embryo lethality repressor.

The developed inducer line may or may not contain selectable markers. Itmay or may not be developed using transgenes that are selectablemarkers.

The following describes typical components for producing an inducerline. Construct A, comprises a non-transmission pollen polynucleotideand an embryo lethality repressor, and a selectable marker gene.Construct B comprises a selectable marker gene and an embryo lethalitygene. To facilitate segregation upon pollination the two constructs canbe located at two different locations in the genome. For furtherefficiency Construct A and Construct B should be unlinked.

Construct A and B can be co-transformed into the inducer line or thetransformation process can be done sequentially, most conveniently withConstruct A first and Construct B second. The initial T₀ inducer plantwill be heterozygous for both constructs, A and B. The expected resultsof the self-pollination of this plant are given in Table 1.

Another way to introduce the constructs into the inducer line would beto cross or breed them into the inducer line. For example, Construct Aand B could be co-transformed into a plant and then bred into an inducerline. For the system to be the most efficient in plants, the embryolethal repressor needs to present with the embryo lethal polynucleotide.Therefore, one may transform with construct containing the embryo lethalrepressor polynucleotide to obtain stably transformed cells and thentransform with construct comprising the embryo lethal polynucleotide.

TABLE 1 Self-pollination of a T₀ plant co-transformed at 2 segregatingloci (genotype (A-B-). T₀ plant production is possible because both theembryo lethal and the embryo lethal repressor are both expressed insomatic embryos and embryogenic callus. Pollen containing Construct A isnon-viable or non-transmissible. Embryos containing Construct B but notConstruct A are non-viable due to the expression of the embryo lethalgene without the embryo lethal repressor gene. The A-BB genotype willhave two doses of the embryo lethal gene and one dose of the embryorepressor gene. Therefore for the greatest efficiency, the expression ofa single repressor polynucleotide should be at a level that will preventlethality of two doses of the embryo lethal polynucleotide. Male GametesAB A- -B -- Female AB non- non- A-BB A-B- Gametes transmissiontransmission Viable Viable of pollen of pollen embryo embryo A- non-non- A-B- A--- transmission transmission Viable Viable of pollen ofpollen embryo embryo but sensitive to selectable marker agent b -B non-non- --BB --B- transmission transmission Embryo Embryo of pollen ofpollen lethal lethal -- non- non- --B- ---- transmission transmissionEmbryo Embryo pollen pollen lethal viable but sensitive to selectablemarker (a and b) agents Construct A = non-transmission pollen + embryolethal repressor + selectable marker gene “a” Construct B = embryolethal + selectable marker “b”

Because there will be viable embryos without both constructs, theembryos or plants from embryos not containing both constructs can beselected against by contacting the tissue with the selectable agent. Ifthe selectable marker is a visual marker, or some other type of marker,this can also be observed and the tissue not containing both markers canbe selected against. Any type of selectable markers can be used. Theremaining plants will either be heterozygous for both constructs (A-B-)or heterozygous for construct A and homozygous for construct B, (A-BB).The result of selfing the A-B-genotype will result in the progeny asseen previously, Table 1. The result of selfing the A-BB genotype willgive you only genotypes with that are homozygous for Construct B.Therefore all the progeny will have the selectable marker b. This is thedesired genotype, A-BB. The plants that are heterozygous for bothconstructs will produce some progeny that do not contain selectablemarker b. The resultant progeny from selfing the A-BB genotypes aredescribed in Table 2.

TABLE 2 Self-pollination of T₁ plants having A-BB genotype. All viableprogeny from selfed A-BB plants will be A-BB plants. Male Gametes AB -BFemale AB non- A-BB Gametes transmission Viable of pollen embryo -B non---BB transmission Embryo of pollen lethal

The desired genotype, A-BB, produces only one viable pollen genotype,-B. When this transgenic inducer line is crossed as a male to a wildtypefemale, this will result in the ablation of all diploid embryos, Table3. However maternal haploid embryos do not inherit the embryo lethalfrom the male parent and are therefore viable. The maternal haploids donot contain the transgenes from Construct A or Construct B.

Even though the maternal haploid embryo does not inherit the DNA fromthe male parent, the pollen carrying the embryo lethal gene will haveone sperm nucleus that fuses with the polar nuclei in the embryo sac tocreate a triploid (3N) endosperm. Therefore the embryo lethal geneeither cannot be expressed in the endosperm or aleurone; or if it isexpressed in these cells it will not kill the haploid embryo.

TABLE 3 Cross of the haploid-induction line, with genotype A-BB, to awildtype female line, ----. Pollen containing Construct A and embryoscontaining Construct B are nonviable due to the expression of the embryolethal without the expression of the embryo lethal repressor. Diploidsinherit Construct B from the male and are nonviable. Maternal haploidsdo not inherit Construct B and are viable. Any maternal embryos thatdevelop normally will be haploid. Male Gametes (inducer line) No male AB-B contribution Female -- non- Embryo -- Gametes transmission lethalMaternal (wildtype) of pollen haploid

The development of an inducer line could also be produced without anyselectable markers. It could also be produced using only one selectablemarker on either construct or with the same selectable marker on bothconstructs. One could use any number of techniques known to one of skillin the art to track and breed for the transgenes. For example progenytests, PCR, molecular markers, or ELISA could be used to track thetransgenes. Also any combination of techniques could be used. Forexample if a first construct=pollen non-transmission+embryo lethalrepressor and a second construct=embryo lethal, quantitative PCR couldbe used to determine which progeny contain which construct and in whatdose, homozygous state or heterozygous state.

Another method of the invention includes co-transformation with thepollen non-transmission and embryo lethal repressor genes on oneconstruct and a selectable marker gene on a second construct. Afterobtaining transformed tissue a second co-transformation can be conductedwith the embryo lethal and a second selectable marker. After transgenicplants are developed the selectable markers can be segregated away fromthe other transgenes.

Another method may be utilized with chemical application or contactacting as the embryo lethal repressor One may be able to obtain plantswith only Construct B if a chemical can be used to repress the embryolethal polynucleotide. For example, one could transform the constructsinto two different plants and then breed both constructs into theinducer line. The chemical that represses the embryo lethality wouldhave to be used for the plant transformed with Construct B until theboth constructs are contained in the same plant. One would then selectfor an inducer line containing the two segregating constructs, A and B.Trait integration through backcrossing is well known in the art.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize include,but are not limited to, Stock 6 and Stock 6 derivatives (Coe, 1959, Am.Nat. 93:381-382; Sarkar and Coe, 1966, Genetics 54:453-464; Sarkar etal, 1972, Development of maternal-haploidy-inducer lines in maize (Zeamays L.) Indian J. Agric. Sci. 42:781-786; Lashermes and Beckert, 1988,Genetic control of maternal haploidy in maize (Zea mays L.) andselection of haploid inducing lines. Theor. Appl. Genet. 76:405-410;Chalyk, S. T. 1994, Properties of maternal haploid maize plants andpotential application to maize breeding. Euphytica 79; 13-18; Bordes, J.R. et al., 1997, Haploidization of maize (Zea mays L.) through inducedgynogenesis assisted by glossy markers and its use in breeding.Agronomie 17:291-297; Eder J. and S. Chalyk, 2002, In vivo haploidinduction in maize. Theor. Appl. Genet. 104:703-708) RWS (Rober,Gordillo, and Geiger, 2005, Maydica 50 (2005) 275-283), KEMS (Deimling,Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224), or KMS andZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk & Chebotar,2000, Plant Breeding 119:363-364), and indeterminate gametophyte (ig)mutation (Kermicle 1969 Science 166:1422-1424). The disclosures of whichare incorporated herein by reference.

Wide hybridization crosses can also be used to produce haploids. Inbarley, this method is sometimes referred to as the bulbosum method(Kasha and Kao, 1970, Nature 225:874-876). This method of haploidproduction occurs due to the elimination of the chromosomes from thepollinating parent.

When an inducer line is used to pollinate a diploid plant, haploidembryos are derived. One sperm nucleus from the pollen fuses with thepolar nuclei in the embryo sac to create a triploid (3N) endosperm. Thetriploid endosperm will contain 2 sets of chromosomes from the femaleand 1 set of chromosomes from the male, which in this case is theinducer line. The haploid embryo contains a single set of chromosomes,which are derived from the female plant.

In the development of haploid maize, Rnj is a commonly used allele forhaploid/diploid screening of mature seeds (Nanda and Chase, 1966. Anembryo marker for detecting monoploids of maize (Zea mays L). Crop Sci.6:213-215; Greenblatt and Bock, 1967. A commercially desirable procedurefor detection of monoploids in maize. J. Hered. 58:9-13). R-njexpression levels have been shown to be correlated with parameters ofkernel maturation (Alexander and Cross, 1983, Grain fill characteristicsof early maize (Zea mays L) strains selected for variable R-njexpression, Euphytica 32:839-844.). More recently, R-scm2 has also beenused (Kato A., 2002, Chromosome doubling of haploid maize seedlingsusing nitrous oxide gas at the flower primordial stage. Plant breeding121:370-377; and Kato A., 2003, Chromosome doubling method, U.S.Publication 2003/0005479).

The R-nj anthocyanin marker gene used to distinguish haploids anddiploids at the mature seed stage is not expressed until late in embryodevelopment. To identify haploid tissue at an earlier stage, haploidembryos can be identified using a transgenic marker, lec1-GFP, which ispresent in the inducer line. The use of lec1-GFP is valuable because thegene allows one to identify the non-haploid embryos at an early stage ofembryo development (U.S. application Ser. No. 09/718,754, U.S. Pat. No.6,486,382). The absence of the GFP marker expression is used to identifyhaploid embryos. Although the haploid embryos can be identified at anearly stage, the GFP system requires a labor intensive screening processto determine which embryos express the GFP marker and which embryos donot express the GFP marker. The use of a lethal marker in an inducerline would only allow haploid embryo formation and thus eliminate theneed for a less efficient screening process. This system would increasethe efficiency of the doubled haploid process.

Various types of systems could be utilized in the inducer line in orderto increase the efficiency of the doubled haploid process: 1)toxicity/antidote systems 2) transcriptional regulator systems 3) genesilencing systems.

An example of an toxicity/antidote system that can be utilized is theBarnase/Barstar system. Barnase is the name of the extracellularribonuclease produced by Bacillus amyloliquefaciens. The inhibitor ofbarnase is called barstar and is produced intracellularly by the sameorganism that secretes barnase. The function of barstar is to protectthe B. amyloliquefaciens from the toxic effects of intracellularbarnase, rendering it inactive (Hartley, R. W. (1989) Barnase andbarstar: Two small proteins to fit and fold together. Trends Biochem.Sci. 14:450-454). Both of these genes have been cloned and sequenced(Hartley, R. W. (1988) Barnase and barstar: Expression of its clonedinhibitor permits expression of a cloned ribonuclease. J. Mol. Biol.202:913-915). It has been shown that barnase expression is lethal toplant cells, and that barstar can protect a plant cell from the effectsof barnase (Mariani et al., (1990) Induction of male-sterility in plantsby a chimeric ribonuclease gene. Nature 347:737-741; Mariani et al.(1992) A chimeric ribonuclease-inhibitor gene restores fertility to malesterile plants. Nature 357:384-387; Beals, T. P. and Goldberg, R. B.(1997) A novel cell ablation strategy blocks tobacco anther dehiscence.Plant Cell 9:1527-1545; Williams et al., (1997) Male sterility throughrecombinant DNA technology. In Pollen Biotechnology for Crop Productionand Improvement, K. R. Shivanna and V. K. Sawhney, eds (Cambridge, UK:Cambridge University Press) pp 237-257). The transmission of a transgenethrough pollen can be prevented by linking the transgene to apollen-lethality gene composed of a cytotoxic gene under the control ofa pollen-specific (gametophyte) promoter (Twell (1995) Diptheriatoxin-mediated cell ablation in developing pollen: vegetative cellablation blocks generative cell migration. Protoplasma 187:144-154;Williams et al., (1997) Male sterility through recombinant DNAtechnology. In Pollen Biotechnology for Crop Production and Improvement,K. R. Shivanna and V. K. Sawhney, eds (Cambridge, UK: CambridgeUniversity Press) pp 237-257). This type of construct can be maintainedbecause it is transmitted through the female. Many pollen specificpromoters have been identified. Other embodiments of the inventioninclude constructs that would deter anther viability or any constructthat would prevent viable pollen transmission. Barstar inhibits thefunction (RNase) of the barnase protein in the haploid-inducer line.Diploid embryos are ablated because the barstar PTU (plant transcriptionunit) is linked to the pollen non-transmission PTU, and thus is notpresent in the diploid embryos to inhibit barnase. The barstar systemcan be improved by optimizing the coding region of the barstar gene. Onemay also optimize the system by increasing the affinity of barstar tobarnase. Other ways of improving the system include increasing theexpression of barstar over the expression level of barnase so that theexpression level is, for example about 1.5×, 2×, or at least 3× thelevel of the expression of barnase. One may have barnase regulated by anembryo preferred promoter, for example led, and have barstar regulatedby a constitutive promoter, for example Ubi. One may also add introns,such as Adh introns, or add enhancers, such as 35S enhancers in order toincrease expression. Introns may be needed in the polynucleotide thatexpresses a toxic product for the purpose of preventing expression ofthe toxic product in the Agrobacterium.

There are many examples of transcriptional regulator systems that can beutilized (Ramos et al. “The tetr family of transcriptional repressors”(2005) Microbiology and Molecular Biology Reviews, vol. 69(2): 326-356).Some examples of regulator families are indicated in Table 4.

TABLE 4 Regulator Families Examples of regulated DNA binding FamilyAction functions motif Position LysR Activator/ Carbon andHelix-turn-helix N-terminal repressor nitrogen metabolism AraC/XylSActivator Carbon Helix-turn-helix C-terminal metabolism, stress responseand pathogenesis TetR Repressor Biosynthesis Helix-turn-helix C-terminalof antibiotics, efflux pumps, osmotic stress, etc. LuxR Activator QuorumHelix-turn-helix C-terminal sensing, biosysnthesis and metabolism, etc.Lac1 Repressor Carbon Helix-turn-helix N-terminal source utilizationArsR Repressor Metal Helix-turn-helix Central resistance IclR Repressor/Carbon Helix-turn-helix N-terminal activator metabolism, efflux pumpsMerR Repressor Resistance Helix-turn-helix N-terminal and detoxificationAsnC Activator/ Amino acid Helix-turn-helix N-terminal repressorbiosynthesis MarR Activator/ Multiple Helix-turn-helix Central repressorantibiotic resistance NtrC Activator Nitrogen Helix-turn-helixC-terminal (EBP) assimilation, aromatic amino acid synthesis, flagella,catabolic pathways, phage response etc. OmpR Activator Heavy metalWinged helix C-terminal and virulence DeoR Repressor SugarHelix-turn-helix N-terminal metabolism Cold Activator Low- RNA bindingVariable shock temperature domain (CSD) resistance GntR RepressorGeneral Helix-turn-helix N-terminal metabolism Crp Activator/ GlobalHelix-turn-helix C-terminal repressor responses, catabolic repressionand anaerobiosis

A well studied regulator system that can be utilized to identifyhaploids early in development is the tet repressor system. Thetetracycline operon system, comprises repressor and operator elements.The operon system is controlled by the presence of tetracycline, andself-regulates the level of expression of tetA and tetR genes. Theproduct of tetA removes tetracycline from the cell. The product of tetRis the repressor protein which binds to the operator elements with aK_(d) of about 10 pM in the absence of tetracycline, thereby blockingexpression or tetA and tetR. The TET repressor can be optimized formaize and the promoter used to stop development of the embryo can bemodified with TET operator sequences.

Another example of a system that can be utilized to identify haploidsearly in development utilizes the lac repressor system (Ulmasov et al.(1997) Plant Mol Biol 35-417-424; Wilde et al. (1992) EMBO J11:1251-1259). This repressor/operator based-system is derived from theprokaryotic operon, E. coli lactose operon. This system controls theactivity of a promoter by placing operator sequences near thetranscriptional start site of a gene such that gene expression from theoperon is inhibited upon the binding of the repressor protein to itscognate operator sequence. However, in the presence of an inducingagent, the binding of the repressor to its operator is inhibited, thusactivating the promoter and enabling gene expression. In the lac system,isopropyl-B-D-thiogalactopyranoside (IPTG) is the commonly used inducingagent, while tetracycline, and/or doxycyline are commonly used inducingagents for the tet system. The lac repressor has been extensivelycharacterized. The lac repressor has a high association constant for itsoperator, and IPTG reduces the affinity of repressor for the operator by300-fold (Barkley & Bourgeois (1980) The Operon, Miller & Reznikoff,Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp 177-220),only 30-fold repression has been reported using the lactose repressor(Ulmasov et al. (1997) Plant Mol Biol 35-417-424). The lac repressor canbe maize optimized and the promoter used to stop development of theembryo can be modified with lac operator sequences.

Gene silencing may also be used in the selective ablation of diploidembryos system. The repression of embryo lethality may be at the RNA orprotein stage for example, anti-sense RNA, hair-pins, and othermechanisms for gene silencing. For example one could have the barnasehairpin polynucleotide driven by a strong promoter such as UBI. Thisconstruct comprising the hairpin can be linked to a construct that doesnot allow pollen fertilization. The other construct could comprise thebarnase polynucleotide driven by the led promoter. This wouldeffectively turn off barnase until the hairpin locus was segregatedaway. Any polynucleotide that prevents normal development of the embryoalong with a corresponding gene silencing construct can be used in thesystem.

Examples of genes that may be driven by a pollen promoter and used toprevent fertile pollen in the invention include DAM (GenBank J01600,Nucleic Acids Res. 11:837-851 (1983); alpha-amylase (GenBank L25805,Plant Physiol. 105(2):759-760 (1994)); D8 (Physiol. Plant 100(3):550-560(1997)); SacB (Plant Physiol. 110(2):355-363 (1996)), lipases andribonucleases; tasselseed2 (ts2) (Calderon-Urrea M. and S. L. Dellaporta(1999) Development 126:435-441); diphtheria toxin A (DTA) (Greenfield etal. Proc. Natl. Acad. Sci. 80:6853 (1983); Palmiter et al., Cell 50:435(1987)).

Examples of pollen specific promoters that can be used include but arenot limited to PG47 (Rogers et al. (1991), Pollen specific cDNA clonesfrom Zea mays, Biochem. Biophys. Acta 1089, 411-413; Allen, R. L., andLonsdale, D. M. (1992) Sequence analysis of three members of the maizepolygalacturonase gene family expressed during pollen development, PlantMol. Biol. 20, 343-345, Allen, R. L., and Lonsdale, D. M. (1993),Molecular characterization of one of the maize polygalacturonase genefamily members which are expressed during late pollen development. ThePlant Journal 3, 261-271, U.S. Pat. No. 5,412,085 and U.S. Pat. No.5,545,546); maize pollen-specific gene Zm13 (Hamilton et al. (1992)Plant Mol. Biol. 18:211-218; Guerrero et al. (1993) Mol. Gen. Genet.224:161-168); microspore-specific promoters such as the apg genepromoter (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)); furtherinclude a sunflower pollen-expressed gene SF3 (Baltz et al. (1992) ThePlant Journal 2:713-721), B. napus pollen specific genes (Arnoldo et al.(1992) J. Cell. Biochem, Abstract No. Y101204). Such promoters are knownin the art or can be discovered by known techniques; see, e.g., Bhallaand Singh (1999) Molecular control of male fertility in Brassica Proc.10^(th) Annual Rapeseed Congress, Canberra, Australia; van Tunen et al.(1990) Pollen-specific chi promoters from petunia: tandem promoterregulation of the chiA gene, Plant Cell 2:393-40; Jeon et al. (1999);and Twell et al. (1993) Activation and developmental regulation of anArabidopsis anther-specific promoter in microspores and pollen ofNicotiana tabacum, Sex. Plant Reprod. 6:217-224.

Many examples of pollen promoters and the polynucleotides used with themto prevent viable pollen can be found in U.S. Pat. No. 6,743,968 and USPublication 2005/0246796 (U.S. application Ser. No. 11/014,071).

Cereal genes whose promoters are associated with early seed and embryodevelopment include lec1 (U.S. Pat. No. 7,122,658), rice glutelin(“GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol 37:107-11;“GluB-1,” Takaiwa et al., 1996, Plant Mol Biol 30:1207-21; Washida etal., 1999, Plant Mol Biol 40:1-12; “Gt3,” Leisy et al., 1990, Plant MolBiol 14:41-50), rice prolamin (Zhou & Fan, 1993, Transgenic Res2:141-6), wheat prolamin (Hammond-Kosack et al., 1993, EMBO J12:545-54), maize zein (Z4, Matzke et al., 1990, Plant Mol Biol14:323-32), and barley B-hordeiis (Entwistle et al., 1991, Plant MolBiol 17:1217-31). “Seed-preferred” promoters include both“seed-specific” promoters (those promoters active during seeddevelopment such as promoters of seed storage proteins) as well as“seed-germinating” promoters (those promoters active during seedgermination). See Thompson et al. (1989) BioEssays 10:108. Suchseed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message), cZ19B1 (maize 19 kDa zein), mi1ps(myo-inositol-1-phosphate synthase); see WO 00/11177 and U.S. Pat. No.6,225,529. Gamma-zein is an endosperm-specific promoter. Globulin-1(Glob-1) is a representative embryo-specific promoter. The maize Glb1gene encodes globulin-1, a major embryo storage protein. (Kriz, A. L.,et al. (1986) Plant Physiol. 82:1069-1075) Glb1 is expressed in thedeveloping maize seed during embryo development. (Belanger, F. C., etal. (1989) Plant Physiol. 91:636-643) The promoter region of Glb1 hasbeen identified, cloned, and introduced into tobacco plants byAgrobacterium-mediated transformation. (Liu, S., et al. (1996) PlantCell Reports 16:158-162) The transformed plants demonstrate that theGlb1 promoter has desirable temporal and tissue specificity. The Glb1promoter is positively regulated by abscisic acid (ABA). (Kriz, A. L.,et al. (1990) Plant Physiol. 92:538-542; Paiva, R., et al., (1994)Planta 192:332-339) Levels of the plant hormone ABA are known tofluctuate under conditions of cold or desiccation. (Himmelbach, A., etal. (1998) Phil. Trans. R. Soc. Lond. 353:1439-1444) Thus, the activityof the Glb1 promoter can be differentially affected. From dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. More examples of seed-specific promoters from monocots include,but are not limited to, maize 15 kDa zein; 22 kDa zein; 27 kDa zein(Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P. and Palau,Jr.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize:Common elements in the 5′ flanking regions among zein and glutelingenes; Plant Sci. 47:95-102 (1986)); gamma-zein; waxy (Kloesgen, R. B.,Gierl, A., Schwarz-Sommer, Z. S. and Saedler, H., Molecular analysis ofthe waxy locus of Zea mays, Mol. Gen. Genet. 203:237-244 (1986));shrunken 1; shrunken 2 (Shaw et al., Plant Phys 98:1214-1216, 1992;Zhong Chen et al., PNAS USA 100:3525-3530, 2003); globulin 1; mZE40-2,also known as Zm-40, U.S. Pat. No. 6,403,862; an ltp2 promoter (Kalla,et al., Plant Journal 6:849-860 (1994); U.S. Pat. No. 5,525,716), cim1promoter (see U.S. Pat. No. 6,225,529); nuc1c (U.S. Pat. No. 6,407,315);etc. See also WO 00/12733 and U.S. Pat. No. 6,528,704, whereseed-preferred promoters from end1 and end2 genes are disclosed.Additional embryo specific promoters are disclosed in Sato et al. (1996)Proc. Natl. Acad. Sci. 93:8117-8122 (rice homeobox, OSH1); andPostma-Haarsma et al. (1999) Plant Mol. Biol. 39:257-71 (rice KNOXgenes). Additional endosperm specific promoters are disclosed in Albaniet al. (1984) EMBO 3:1405-15; Albani et al. (1999) Theor. Appl. Gen.98:1253-62; Albani et al. (1993) Plant J. 4:343-55; Mena et al. (1998)The Plant Journal 116:53-62 (barley DOF); Opsahl-Ferstad et al. (1997)Plant J 12:235-46 (maize Esr); and Wu et al. (1998) Plant CellPhysiology 39:885-889 (rice GluA-3, GluB-1, NRP33, RAG-1).

Examples of constitutive promoters include the 1′- or 2′-promoter ofAgrobacterium tumefaciens (see, e.g., O'Grady (1995) Plant Mol. Biol.29:99-108). Other plant promoters include the ribulose-1,3-bisphosphatecarboxylase small subunit promoter, the phaseolin promoter, alcoholdehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant Mol.Biol. 31:897-904), sucrose synthase promoters, α-tubulin promoters,actin promoters, such as the Arabidopsis actin gene promoter (see, e.g.,Huang (1997) Plant Mol. Biol. 1997 33:125-139), cab, PEPCase, R genecomplex, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol.33:125-139 (1996)), Cat3 from Arabidopsis (Zhong et al., Mol. Gen.Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrierprotein desaturase from Brassica napes (Solocombe et al. (1994) PlantPhysiol. 104:1167-1176), GPc1 from maize (Martinez et al. (1989) J. Mol.Biol 208:551-565), Gpc2 from maize (Manjunath et al. (1997), Plant Mol.Biol. 33:97-112), and other transcription initiation regions fromvarious plant genes known to those of skill. See also Holtorf (1995)“Comparison of different constitutive and inducible promoters for theoverexpression of transgenes in Arabidopsis thaliana,” Plant Mol. Biol.29:637-646. The promoter sequence from the E8 gene (see, Deikman andFischer (1988) EMBO J 7:3315) and other genes can also be used, alongwith promoters specific for monocotyledonous species (e.g., McElroy D.,et al. (1994.) Foreign gene expression in transgenic cereals, TrendsBiotech. 12:62-68). Other constitutive promoters include, for example,the core promoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Yet, otherconstitutive promoters include, for example, U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611.

In addition to the promoters noted herein, promoters of bacterial originwhich operate in plants can be used in the invention. They include,e.g., the octopine synthase promoter, the nopaline synthase promoter andother promoters derived from Ti plasmids. See, Herrera-Estrella et al.(1983) Nature 303:209. Viral promoters can also be used. Examples ofviral promoters include the 35S and 19S RNA promoters of cauliflowermosaic virus (CaMV). See, Odell et al., (1985) Nature 313:810; and,Dagless (1997) Arch. Virol. 142:183-191. Other examples of constitutivepromoters from viruses which infect plants include the promoter of thetobacco mosaic virus; cauliflower mosaic virus (CaMV) 19S and 35Spromoters or the promoter of Figwort mosaic virus, e.g., the figwortmosaic virus 35S promoter (see, e.g., Maiti (1997) Transgenic Res.6:143-156), etc. Alternatively, novel promoters with usefulcharacteristics can be identified from any viral, bacterial, or plantsource by methods, including sequence analysis, enhancer or promotertrapping, and the like, known in the art.

Tissue-preferred (tissue-specific) promoters and enhancers can beutilized to target enhanced gene expression within a particular planttissue. Tissue-preferred (tissue-specific) promoters include, e.g.,those described in Yamamoto et al. (1997) Plant J. 12(2):255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al.(1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535;Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakerexpression or stronger expression. A tissue-specific promoter can driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein, a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but can also leadto some expression in other tissues as well.

In certain embodiments, leaf specific promoters can be used, e.g.,pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize),cab-m1 Ca+2 promoter from maize, the Arabidopsis thaliana myb-relatedgene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS)promoters (e.g., the tomato RBCS1, RBCS2 and RBCS3A genes, which areexpressed in leaves and light-grown seedlings, while RBCS1 and RBCS2 areexpressed in developing tomato fruits, and/or a ribulose bisphosphatecarboxylase promoter which is expressed almost exclusively in mesophyllcells in leaf blades and leaf sheaths at high levels, etc.), and thelike. See, e.g., Matsuoka et al., (1993) Tissue-specific light-regulatedexpression directed by the promoter of a C4 gene, maize pyruvate,orthophosphate dikinase, in a C3 plant, rice, PNAS USA 90(20):9586-90;(2000) Plant Cell Physiol. 41(1):42-48; (2001) Plant Mol. Biol.45(1):1-15; Shiina, T. et al., (1997) Identification of PromoterElements involved in the cytosolic Ca+2 mediated photoregulation ofmaize cab-m1 expression, Plant Physiol. 115:477-483; Casal (1998) PlantPhysiol. 116:1533-1538; Li (1996) FEBS Lett. 379:117-121; Busk (1997)Plant J. 11:1285-1295; and, Meier (1997) FEBS Lett. 415:91-95; and,Matsuoka (1994) Plant J. 6:311-319. Other leaf-specific promotersinclude, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265;Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994)Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J.3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; andMatsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

In certain embodiments, senescence specific promoters can be used (e.g.,a tomato promoter active during fruit ripening, senescence andabscission of leaves, a maize promoter of gene encoding a cysteineprotease, and the like). See, e.g., Blume (1997) Plant J. 12:731-746;Griffiths et al., (1997) Sequencing, expression pattern and RFLP mappingof a senescence-enhanced cDNA from Zea Mays with high homology tooryzain gamma and aleurain, Plant Mol. Biol. 34(5):815-21; Zea mayspartial see1 gene for cysteine protease, promoter region and 5′ codingregion, Genbank AJ494982; Kleber-Janke, T. and Krupinska, K. (1997)Isolation of cDNA clones for genes showing enhanced expression in barleyleaves during dark-induced senescence as well as during senescence underfield conditions, Planta 203(3):332-40; and, Lee, R H et al., (2001)Leaf senescence in rice plants: cloning and characterization ofsenescence up-regulated genes, J. Exp. Bot. 52(358):1117-21.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue (see, e.g., EMBO J. 8(2):343-350).The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showedsimilar characteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

The use of temporally-acting promoters is also contemplated by thisinvention. For example, promoters that act from 0-25 days afterpollination (DAP), 4-21, 4-12, or 8-12 DAP can be selected, e.g.,promoters such as cim1 and ltp2. Promoters that act from 0-14 days afterpollination can also be used, such as SAG12 (See WO 96/29858, Richard M.Amasino, published 3 Oct. 1996) and ZAG1 or ZAG2 (See R. J. Schmidt, etal., Identification and Molecular Characterization of ZAG1, the MaizeHomolog of the Arabidopsis Floral Homeotic Gene AGAMOUS, Plant-Cell5(7):729-37 (July 1993)). Other useful promoters include maize zag2. 1,Zap (also known as ZmMADS; U.S. patent application Ser. No. 10/387,937;WO 03/078590); and the maize tb1 promoter (see also Hubbarda et al.,Genetics 162:1927-1935, 2002).

Shoot-preferred promoters include, shoot meristem-preferred promoterssuch as promoters disclosed in Weigel et al. (1992) Cell 69:853-859;Accession No. AJ131822; Accession No. Z71981; Accession No. AF059870,the ZAP promoter (U.S. patent application Ser. No. 10/387,937), themaize promoter (Wang et al. (1999) Nature 398:236-239, andshoot-preferred promoters disclosed in McAvoy et al. (2003) Acta Hort.(ISHS) 625:379-385.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed, or will be transcribed at alevel lower than in an induced state. The inducer can be a chemicalagent, such as a metabolite, growth regulator, herbicide or phenoliccompound, or a physiological stress directly imposed upon the plant suchas cold, drought, heat, salt, toxins. Plant promoters which areinducible upon exposure to plant hormones, such as auxins, can be used.For example, the invention can use the auxin-response elements E1promoter subsequence (AuxREs) from the soybean (Glycine max L.) (Liu(1997) Plant Physiol. 115:397-407); the auxin-responsive ArabidopsisGST6 promoter (also responsive to salicylic acid and hydrogen peroxide)(Chen (1996) Plant J. 10:955-966); the auxin-inducible parC promoterfrom tobacco; a plant biotin response element (Streit (1997) Mol. PlantMicrobe Interact. 10:933-937); and the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902). Plantpromoters which are inducible upon exposure to chemical reagents whichcan be applied to the plant, such as herbicides or antibiotics, are alsoused to express polynucleotides. The promoter can be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. For example, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, can beused (De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. An ACC synthase coding sequence or RNA configuration can alsobe under the control of, e.g., tetracycline-inducible andtetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol.Gen. Genet. 227:229-237; U.S. Pat. Nos. 5,814,618 and 5,789,156; and,Masgrau (1997) Plant J. 11:465-473 (describing transgenic tobacco plantscontaining the Avena sativa L. (oat) arginine decarboxylase gene with atetracycline-inducible promoter); or, a salicylic acid-responsiveelement (Stange (1997) Plant J. 11:1315-1324. Other chemical-induciblepromoters are known in the art and include, but are not limited to, themaize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257).

Examples of inducible regulatory elements include a metallothioneinregulatory element, a copper-inducible regulatory element, or atetracycline-inducible regulatory element, the transcription from whichcan be effected in response to divalent metal ions, copper ortetracycline, respectively (Furst et al., Cell 55:705-717, 1988; Mett etal., Proc. Natl. Acad. Sci., USA 90:4567-4571, 1993; Gatz et al., PlantJ. 2:397-404, 1992; Roder et al., Mol. Gen. Genet. 243:32-38, 1994).Inducible regulatory elements also include an ecdysone regulatoryelement or a glucocorticoid regulatory element, the transcription fromwhich can be effected in response to ecdysone or other steroid(Christopherson et al., Proc. Natl. Acad. Sci., USA 89:6314-6318, 1992;Schena et al., Proc. Natl. Acad. Sci., USA 88:10421-10425, 1991; U.S.Pat. No. 6,504,082); a cold responsive regulatory element or a heatshock regulatory element, the transcription of which can be effected inresponse to exposure to cold or heat, respectively (Takahashi et al.,Plant Physiol. 99:383-390, 1992); the promoter of the alcoholdehydrogenase gene (Gerlach et al., PNAS USA 79:2981-2985 (1982); Walkeret al., PNAS 84(19):6624-6628 (1987)), inducible by anaerobicconditions; and the light-inducible promoter derived from the pea rbcSgene or pea psaDb gene (Yamamoto et al. (1997) Plant J. 12(2):255-265);a light-inducible regulatory element (Feinbaum et al., Mol. Gen. Genet.226:449, 1991; Lam and Chua, Science 248:471, 1990; Matsuoka et al.(1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590; Orozco et al. (1993)Plant Mol. Bio. 23(6):1129-1138), a plant hormone inducible regulatoryelement (Yamaguchi-Shinozaki et al., Plant Mol. Biol. 15:905, 1990;Kares et al., Plant Mol. Biol. 15:225, 1990), and the like. An inducibleregulatory element also can be the promoter of the maize In2-1 or In2-2gene which responds to benzenesulfonamide herbicide safeners (Hershey etal., Mol. Gen. Gene 227:229-237, 1991; Gatz et al., Mol. Gen. Genet.243:32-38, 1994), and the Tet repressor of transposon Tn10 (Gatz et al.,Mol. Gen. Genet. 227:229-237, 1991). Stress inducible promoters includesalt/water stress-inducible promoters such as P5CS (Zang et al. (1997)Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a(Hajela et al. (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm etal. (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet et al. (1998)FEBS Lett. 423-324-328), ci7 (Kirch et al. (1997) Plant Mol Biol.33:897-909), ci21A (Schneider et al. (1997) Plant Physiol. 113:335-45);drought-inducible promoters, such as, Trg-31 (Chaudhary et al. (1996)Plant Mol. Biol. 30:1247-57), rd29 (Kasuga et al. (1999) NatureBiotechnology 18:287-291); osmotic inducible promoters, such as Rab17(Vilardell et al. (1991) Plant Mol. Biol. 17:985-93) and osmotin(Raghothama et al. (1993) Plant Mol Biol 23:1117-28); and heat induciblepromoters, such as heat shock proteins (Barros et al. (1992) Plant Mol.19:665-75; Marrs et al. (1993) Dev. Genet. 14:2741), smHSP (Waters etal. (1996) J. Experimental Botany 47:325-338), and the heat-shockinducible element from the parsley ubiquitin promoter (WO 03/102198).Other stress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808and U.S. Publication No. 2003/0217393) and rd29a (Yamaguchi-Shinozaki etal. (1993) Mol. Gen. Genetics 236:331-340). Certain promoters areinducible by wounding, including the Agrobacterium pmas promoter(Guevara-Garcia et al. (1993) Plant J. 4(3):495-505) and theAgrobacterium ORF13 promoter (Hansen et al., (1997) Mol. Gen. Genet.254(3):337-343).

The expression cassette used in the invention can include, at the 3′terminus of the heterologous nucleotide sequence of interest, atranscriptional and translational termination region functional inplants. The termination region can be native with the promoternucleotide sequence of the present invention, can be native with the DNAsequence of interest, or can be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.The 3′ terminus of the pinII—(potato proteinase inhibitor) can be used.See Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996)Nature Biotechnology 14:494-498. For other 3′ terminus sequences seealso, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshiet al. (1987) Nucleic Acid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. (1989) Proc. Nat Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al. (1986); MDMV leader (Maize Dwarf Mosaic Virus), Virology154:9-20; human immunoglobulin heavy-chain binding protein (BiP),Macejak et al. (1991) Nature 353:90-94; untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV), Gallie etal. (1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al. (1991) Virology 81:382-385. Seealso Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

Any type of transformation can be utilized to obtain the selectiveablation inducer line. Transformation protocols as well as protocols forintroducing nucleotide sequences into plants may vary depending on thetype of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing nucleotide sequencesinto plant cells and subsequent insertion into the plant genome includemicroinjection (Crossway et al. (1986) Biotechniques 4:320-334),electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606, Agrobacterium-mediated transformation (Townsend et al.,U.S. Pat. No. 5,563,055), direct gene transfer (Paszkowski et al. (1984)EMBO J. 3:2717-2722), and ballistic particle acceleration (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926). Also see Weissinger et al.(1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37 (onion); Christou et al. (1988) PlantPhysiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Ishida et al. (1996) Nature Biotechnology 14:745-750; U.S. Pat.No. 5,731,179; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,641,664; andU.S. Pat. No. 5,981,840 (maize via Agrobacterium tumefaciens); thedisclosures of which are herein incorporated by reference.

In planta Agrobacterium transformation is disclosed in the following:Bechtold, N., J. Ellis, G. Pelletier (1993) C. R., Acad Sci Paris LifeSci 316:1194-1199; Bechtold, N., B. et al. (2000) Genetics155:1875-1887; Bechtold, N. and G. Pelletier (1998) Methods Mol Biol.82:259-266; Chowrira, G. M., V. Akella, and P. F. Lurquin (1995) Mol.Biotechnol. 3:17-23; Clough, S. J., and A. F. Bent (1998) Plant J.16:735-743; Desfeux, C., S. J. Clough, and A. F. Bent. (2000) PlantPhysiol. 123: 895-904; Feldmann, K. A., and M. D. Marks (1987) Mol. Gen.Genet. 208:1-9; Hu C.-Y., and L. Wang. (1999) In Vitro Cell Dev.Biol.-Plant 35:417-420; Katavic, V. G. W. Haughn, D. Reed, M. Martin, L.Kunst (1994) Mol. Gen. Genet. 245: 363-370; Liu, F., et al. (1998) ActaHort 467:187-192; Mysore, K. S., C. T. Kumar, and S. B. Gelvin (2000)Plant J. 21:9-16; Touraev, A., E. Stoger, V. Voronin, and E.Heberle-Bors (1997) Plant J. 12:949-956; Trieu, A. T. et al. (2000)Plant J. 22:531-541; Ye, G. N. et al. (1999) Plant J. 19:249-257; Zhang,J U. et al. (2000) Chem Biol. 7:611-621. The disclosures of the aboveare herein incorporated by reference.

Various types of plant tissue can be used for transformation such asembryo cells, meristematic cells, leaf cells, or callus cells derivedfrom embryo, leaf or meristematic cells. However, anytransformation-competent cell or tissue can be used. Various methods forincreasing transformation frequency may also be employed. Such methodsare disclosed in WO 99/61619; WO 00/17364; WO 00/28058; WO 00/37645;U.S. Ser. No. 09/496,444; WO 00/50614; US01/44038; and WO 02/04649. Thedisclosures of the above are herein incorporated by reference.

Transformation of maize can follow a well-established bombardmenttransformation protocol used for introducing DNA into the scutellum ofimmature maize embryos (See, e.g., Tomes et al., Direct DNA Transferinto Intact Plant Cells Via Microprojectile Bombardment. pp. 197-213 inPlant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L.Gamborg and G. C. Phillips. Springer-Verlag Berlin Heidelberg N.Y.,1995.). Cells are transformed by culturing maize immature embryos(approximately 1-1.5 mm in length) onto medium containing N6 salts,Erikkson's vitamins, 0.69 g/l proline, 2 mg/l 2,4-D and 3% sucrose.After 4-5 days of incubation in the dark at 28° C., embryos are removedfrom the first medium and cultured onto similar medium containing 12%sucrose. Embryos are allowed to acclimate to this medium for 3 h priorto transformation. The scutellar surface of the immature embryos istargeted using particle bombardment. Embryos are transformed using thePDS-1000 Helium Gun from Bio-Rad at one shot per sample using 650PSIrupture disks. DNA delivered per shot averages at 0.1667 μg. Followingbombardment, all embryos are maintained on standard maize culture medium(N6 salts, Erikkson's vitamins, 0.69 g/l proline, 2 mg/l 2,4-D, 3%sucrose) for 2-3 days and then transferred to N6-based medium containinga selective agent. Plates are maintained at 28° C. in the dark and areobserved for colony recovery with transfers to fresh medium every two tothree weeks. Recovered colonies and plants are scored based on theselectable or screenable phenotype imparted by the marker gene(s)introduced (i.e. herbicide resistance, fluorescence or anthocyaninproduction), and by molecular characterization via PCR and Southernanalysis.

Transformation of maize can also be done using the Agrobacteriummediated DNA delivery method, as described by U.S. Pat. No. 5,981,840with the following modifications. Agrobacteria are grown to the logphase in liquid minimal A medium containing 100 μM spectinomycin.Embryos are immersed in a log phase suspension of Agrobacteria adjustedto obtain an effective concentration of 5×10⁸ cfu/ml. Embryos areinfected for 5 minutes and then co-cultured on culture medium containingacetosyringone for 7 days at 20° C. in the dark. After 7 days, theembryos are transferred to standard culture medium (MS salts with N6macronutrients, 1 mg/L 2,4-D, 1 mg/L Dicamba, 20 g/L sucrose, 0.6 g/Lglucose, 1 mg/L silver nitrate, and 100 mg/L carbenicillin) with aselective agent. Plates are maintained at 28° C. in the dark and areobserved for colony recovery with transfers to fresh medium every two tothree weeks. Recovered colonies and plants are scored based on theselectable or screenable phenotype imparted by the marker gene(s)introduced (i.e. herbicide resistance, fluorescence or anthocyaninproduction), and by molecular characterization via PCR and Southernanalysis.

A selectable marker can be utilized in the recovery of transformedcells. Marker genes include genes encoding antibiotic resistance, suchas those encoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium (the activeingredient in BASTA™), bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). For example, an herbicide resistancepolynucleotide encoding glyphosate N-acetyltransferase (GAT) could beutilized with the selectable agent glyphosate. See PCT publicationWO02/36782 and U.S. application Ser. No. 10/427,692. The PAT(phosphinothricin acetyltansferase) polynucleotide could be used forresistance to phosphinothricin (DeBlock et al., 1987, Engineeringherbicide resistance in plant by expression of a detoxifying enzyme,EMBO J. 6:2513-2518). Another example is an ALS (acetolactate synthase)polynucleotide for resistance to imidazolines (Sathasivan et al., 1990,Nucleotide sequence of a mutant acetolactate synthase gene from animidazolinone-resistant Arabidopsis thaliana var, Columbia, NucleicAcids Res. 18:2188). Additional selectable markers include phenotypicmarkers such as β-galactosidase and fluorescent proteins such as greenfluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein(CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al.(2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP™from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). Foradditional selectable markers, see generally, Yarranton (1992) Curr.Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad.Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference. The above list of selectable marker genes is not meant tobe limiting. Any selectable marker gene can be used in the presentinvention.

Once haploid embryos have been identified the haploid cells, haploidembryos, haploid seeds, haploid seedlings or haploid plants can betreated with a chromosome doubling agent. Homozygous plants can beregenerated from haploid cells by contacting the haploid cells, such ashaploid embryo cells, with chromosome doubling agents. The haploid cellsmay come in contact with the doubling agent at the time of pollination,anytime after pollination, typically 6 hours to 21 days afterpollination, 6 hours to 15 days after pollination, at the mature seedstage, at the seedling stage, or at the plant stage. The haploid embryomay come in contact with the doubling agent when one sperm nucleus froma pollen grain fuses with the polar nuclei in the embryo sac to create atriploid (3N) endosperm (when the haploid embryo is formed), anytimeafter the pollination, typically 6 hours to 21 days after pollination, 6hours to 15 days after pollination, or at the mature seed stage. Thehaploid embryo may be isolated. It may be contained within the kernel,ovule, or seed. It may also be on the ear in the case of corn, or on thespike as in the case of other grains such as wheat. The ear comprisingthe haploid embryo may be on the plant or isolated from the plant. Theear also may be sectioned. After chromosome doubling, the doubledhaploid embryo will contain 2 copies of maternally derived chromosomes.The efficiency of the process for obtaining doubled haploid plants fromhaploid embryos may be greater than 10%, 20%, 30%, 50%, 60%, 70%, 80%,or 90%.

Methods of chromosome doubling are disclosed in Antoine-Michard, S. etal., Plant cell, tissue organ cult., Cordrecht, the Netherlands, KluwerAcademic Publishers, 1997, 48(3):203-207; Kato, A., Maize GeneticsCooperation Newsletter 1997, 36-37; and Wan, Y. et al., TAG, 1989,77:889-892. Wan, Y. et al., TAG, 1991, 81:205-211. The disclosures ofwhich are incorporated herein by reference. Typical methods involvecontacting the cells with colchicine, anti-microtubule agents oranti-microtubule herbicides, pronamide, nitrous oxide, or any mitoticinhibitor to create homozygous doubled haploid cells. The amount ofcolchicine used in medium is generally 0.01%-0.2% or approximately 0.05%or APM (5-225 μM). The amount of pronamide in medium is approximately0.5-20 μM. Examples of known mitotic inhibitors are included but notlimited to those indicated in Table 5. Other agents may be used with themitotic inhibitors to improve doubling efficiency. Such agents may bedimethyl sulfoxide (DMSO), adjuvants, surfactants, and the like.

TABLE 5 Common Name/ Trade name CAS IUPAC Colchicine and ColchicineDerivatives colchicine/ (S)—N-(5,6,7,9-tetrahydro-1,2,3,10-acetyltrimethylcol- tetramethoxy-9-oxobenzo (a) chicinic acidheptalen-7-yl) acetamide colchicine derivatives Carbamates Carbetamide(R)-1- (2R)—N-ethyl-2- (ethylcarbamoyl)ethyl[[(phenylamino)carbonyl]oxy]propanamide carbanilate chloroprophampropham Benzamides Pronamide/ 3,5-dichloro-N-(1,1-3,5-dichloro-N-(1,1-dimethyl-2- propyzamide dimethylpropynyl)benzamidepropynyl)benzamide tebutam Benzoic Acids Chlorthal dimethyl (DCPA),Dicamba/dianat/ 3,6-dichloro-o-anisic acid 3,6-dichloro-2-methoxybenzoicdisugran (dicamba- acid methyl) (BANVEL, CLARITY) Dinitroanilinechromosome doubling agents benfluralin/benefin/ N-butyl-N-ethyl-α,α,α-N-butyl-N-ethyl-2,6-dinitro-4- (BALAN) trifluoro-2,6-dinitro-p-(trifluoromethyl)benzenamine toluidine butralin (RS)—N-sec-butyl-4-tert-4-(1,1-dimethylethyl)-N-(1- butyl-2,6-dinitroaniline methylpropyl)-2,6-dinitrobenzenamine chloralin dinitramine N1,N1-diethyl-2,6-dinitro-N3,N3-diethyl-2,4-dinitro-6- 4-trifluoromethyl-m- (trifluoromethyl)-1,3-phenylenediamine benzenediamine ethalfluralin N-ethyl-α,α,α-trifluoro-N-N-ethyl-N-(2-methyl-2-propenyl)- (Sonalan) (2-methylallyl)-2,6-dinitro-2,6-dinitro-4- p-toluidine (trifluoromethyl)benzenamine fluchloralinN-(2-chloroethyl)-2,6- N-(2-chloroethyl)-2,6-dinitro-N-dinitro-N-propyl-4- propyl-4- (trifluoromethyl)aniline(trifluoromethyl)benzenamine or N-(2-chloroethyl)-α,α,α-trifluoro-2,6-dinitro-N- propyl-p-toluidine isopropalin4-isopropyl-2,6-dinitro- 4-(1-methylethyl)-2,6-dinitro-N,N-N,N-dipropylaniline dipropylbenzenamine methalpropalinα,α,α-trifluoro-N-(2- N-(2-methyl-2-propenyl)-2,6-methylallyl)-2,6-dinitro-N- dinitro-N-propyl-4- propyl-p-toluidine(trifluoromethyl)benzenamine nitralin 4-methylsulfonyl-2,6-4-(methylsulfonyl)-2,6-dinitro-N,N- dinitro-N,N-dipropylanilinedipropylbenzenamine oryzalin (SURFLAN) 3,5-dinitro-N4,N4-4-(dipropylamino)-3,5- dipropylsulfanilamide dinitrobenzenesulfonamidependimethalin N-(1-ethylpropyl)-2,6- N-(1-ethylpropyl)-3,4-dimethyl-2,6-(PROWL) dinitro-3,4-xylidine dinitrobenzenamine prodiamine5-dipropylamino-α,α,α- 2,4-dinitro-N3,N3-dipropyl-6-trifluoro-4,6-dinitro-o- (trifluoromethyl)-1,3- toluidine benzenediamineor 2,6-dinitro-N1,N1-dipropyl- 4-trifluoromethyl-m- phenylenediamineprofluralin N-cyclopropylmethyl- N-(cyclopropylmethyl)-2,6-dinitro-α,α,α-trifluoro-2,6-dinitro- N-propyl-4- N-propyl-p-toluidine(trifluoromethyl)benzenamine or N-cyclopropylmethyl-2,6-dinitro-N-propyl-4- trifluoromethylaniline trifluralin (TREFLAN,α,α,α-trifluoro-2,6-dinitro- 2,6-dinitro-N,N-dipropyl-4- TRIFIC,TRILLIN) N,N-dipropyl-p-toluidine (trifluoromethyl)benzenaminePhosphoroamidates APM (Amiprofos methyl); amiprophos- methyl ButamifosO-ethyl O-6-nitro-m-tolyl O-ethyl O-(5-methyl-2-nitrophenyl) (RS)-sec-(1- butylphosphoramidothioate methylpropyl)phosphoramidothioatePyridines Dithiopyr Thiazopyr methyl 2-difluoromethyl-5- methyl2-(difluoromethyl)-5-(4,5- (4,5-dihydro-1,3-thiazol-2-dihydro-2-thiazolyl)-4-(2- yl)-4-isobutyl-6-methylpropyl)-6-(trifluoromethyl)-3- trifluoromethylnicotinatepyridinecarboxylate

The chromosome doubling agent may come in contact with the embryo atvarious times. If the embryo is isolated the doubling agent may come incontact immediately after isolation and before germination. If theembryo is contained within the seed, it may come in contact with thedoubling agent anytime after pollination and before dry down. The embryowhether it is isolated or not may come in contact with the doublingagent any time between 6 hours after pollination and 21 days afterpollination. The duration of contact between the chromosomal doublingagent may vary. Contact may be from less than 24 hours to about a week.The duration of contact is generally from about 24 hours to 2 days.

Methods provided may or may not go through a callus formation stage. Thehaploid embryos may be placed on a “non-callus” promoting medium. Theterm “non-callus promoting medium” refers to a medium that does notsupport proliferation of dedifferentiated masses of cells or tissue. Apreferred “non-callus promoting medium” is used for embryo rescue,containing typical salt and vitamin formulations well known in the art.Such embryo rescue, or embryo culture, media contain little or no auxin[for review see Raghaven, V., 1966. Biol. Rev. 41:1-58]. Embryomaturation medium also represents another preferred “non-calluspromoting medium”. Embryo maturation medium is used to promotedevelopment of in vitro cultured embryos, preventing precociousgermination, and typically contain standard salt/vitamin formulations(depending on the species), increased sugar levels and/or exogenouslyadded abscisic acid, with little or no auxin. Another type of medium isused for shoot culture, or multiple shoot proliferation. Thismultiple-shoot medium can again contain little or reduced auxin, butinstead contain elevated levels of cytokinin that promote meristemproliferation and growth.

An auxin is defined as an endogenous plant hormone such as indole aceticacid (IAA), derivatives of IAA such as indole-3-buteric acid, as well ascompounds with auxin-like activity such as 2,4-D; picloram; dicamba;3,4-D; 2,4,5-T and naphthalene acetic acid (NAA).

A cytokinin is defined as a naturally occurring plant hormone such as2-isopentynel adenine (21P), zeatin and dihydrozeatin, or a syntheticcompound with cytokinin-like activity such as kinetin and BAP(beynzylaminopurine).

Haploid cells from embryos, seeds, plants, etc. can be identified byseveral methods, such as, by chromosomal counts, measuring the length ofguard cells, or by use of a Flow Cytometer.

Molecular markers or quantitative PCR can be used to determine if atissue or plant is made of doubled haploid cells or is made of diploidcells (cells obtained through normal pollination).

Haploid embryos which are derived by any of the above techniques can becultured to regenerate a whole plant. Such techniques are called embryorescue. Embryo rescue media can comprise certain phytohormones andenergy sources or just energy sources. The growth medium may alsocontain a selection agent such as a biocide and/or herbicide. Thisselection agent can be used to indicate a marker which has beenintroduced through the transformation process. For transformation andregeneration of maize see, Gordon-Kamm et al., The Plant Cell 2:603-618(1990).

The methods provided can be practiced with any plant. Such plantsinclude but are not limited to Zea mays (also identified as corn ormaize), soybean, oilseed Brassica, alfalfa, rice, rye, sorghum,sunflower, tobacco, potato, peanuts, cotton, sweet potato, cassava,sugar beets, tomato, oats, barley, and wheat.

Generation of embryos into plants is well known in the art. Embryorescue techniques can be used to generate immature doubled haploidembryos into plants (Recent Research Developments in Genetics &Breeding. Vol. 1, Part II, 287-308 2004). The disclosure of which isherein incorporated by reference.

The temperature at which the methods can be performed can vary. Themethods provided can be practiced at any temperature that does not killa plant cell or plant or from about 16 degrees Celsius to 32 degreesCelsius.

Provided are methods of producing haploid embryos and non-viable diploidembryos on a maize plant. The method includes but is not limited toproducing about 3% or greater, about 5% or greater, or about 10% orgreater viable haploid embryos. For calculating the percentage, thetotal number of embryos is determined by adding the number of haploidembryos to the number of embryos that did not develop properly. Thenon-viable diploid embryos may be selected against any time afterpollination. The method includes but is not limited to selection at 7-15days, 10-21 days, or at the time the haploid seed is fully mature, forexample during or after dry down.

Provided are methods of selecting for haploid maize embryos bypollinating a maize ear with the pollen from an inducer maize line. Theinducer line has a gene that is expressed in the embryo and can belethal. At another location in the genome the inducer maize line has agene that prevents pollen transmission. The gene that prevents pollentransmission is closely linked to a gene that when expressed in theembryo inhibits the lethality of the gene at the first location. Thus,when the maize ear is pollinated by this inducer line there is notransmission of pollen that contains the gene that prevents the lethaleffects of the lethal gene. Only pollen with the gene that is lethal toembryo development is transmitted. When this pollen is used and normalfertilization occurs resulting in a diploid embryo, the embryo will notdevelop because of the transmission of the lethal gene. When this pollenis used and irregular fertilization occurs it results in a haploidembryo. This haploid embryo will continue to develop. Because of theablation of the diploid embryos, haploid embryos will easily be selectedat an early stage of development. Having the lethal gene and theinhibitor gene at different locations in the genome, for example havingthe genes unlinked, allows the genes to segregate so that an adequateamount of pollen comprising the lethal gene is produced. The gene thatprevents pollen transmission is closely linked to, or adjacent to, thegene that inhibits embryo ablation.

Another method provided is the development of an improved inducer line,called a selective ablation inducer line. A maize inducer line can betransformed with two different expression cassettes. A first expressioncassette includes a polynucleotide that when expressed in the embryo islethal to the embryo or prevents the embryo from developing normally. Asecond expression cassette includes a polynucleotide that preventsviable or transmissible pollen from developing and a polynucleotide thatwhen expressed in the embryo prevents death or abnormal growth thatwould be caused by the polynucleotide on the first expression cassette.The transformation process can be by various methods for exampleparticle bombardment or agrobacterium infection. The transformationprocess with the two cassettes may be done simultaneously orsequentially with the cassette comprising the polynucleotide to preventembryo lethality being introgressed into the plant cell first. Theexpression cassettes need to segregate in the gamete, therefore it ispreferred that the two expression cassettes not be tightly linked. Aspart of the method, any maize line may be transformed with the first, orfirst and second expression cassette. The first and/or second expressioncassettes may be used to transform one or two maize plants. Theexpression cassettes can then be transferred simultaneously orsequentially into a maize inducer line by crossing. The method caninclude backcrossing one or all trangenes into a maize inducer line. Therepressor could be replaced and segregated out.

In any of the methods disclosed the inhibition described can be byvarious mechanisms. For example, the transcription of the lethalpolynucleotide may be prevented. This type of inhibition could beachieved by using a tet repressor polynucleotide that expresses aprotein that binds to the lethal gene and inhibits expression. Othertypes of prevention of lethality may be at the RNA or protein stage forexample, anti-sense RNA, hair-pins, and other mechanisms for genesilencing.

In any of these methods and products, the gene that prevents pollentransmission can be a lethal gene with a pollen specific promoter. Forexample the gene can express alpha-amylase (Gene Bank L25805), PlantPhysiology 105(2):759-760 (1994)) and it can be controlled with a PG47promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No. 5,545,546; Plant J.3(2): 261-271 (1993)). In any of these methods and products theexpression of the polynucleotide that causes ablation and thepolynucleotide that inhibits ablation can be controlled by various typesof promoters. For example the promoter may be a constitutive promoter,an inducible promoter, or a tissue preferred promoter such as an embryopreferred promoter. It would be most efficient if the promoter thatdrives the polynucleotide that causes ablation of the embryo would notexpress in endosperm. Or if the promoter is expressed in the endospermthe expression would be low enough that one could still determine adifference in growth between the diploid seed (normal fertilization) andthe haploid seed. An example of embryo preferred promoter is led.

The invention disclosed includes a maize inducer line comprising the twoexpression cassettes as described.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

gat—genes encoding glyphosate N-acetyltransferase (GAT). See PCTpublication WO02/36782 and U.S. application Ser. No. 10/427,692.

lec1—indicates a leafy cotyledon 1 transcriptional activatorpolynucleotide. See U.S. patent application Ser. No. 09/435,054. lec1promoter is characterized in U.S. Pat. No. 7,122,658.

moCah—is a maize optimized gene that encodes for the Myrotheciurnverrucaria cyanamide hydratase protein [CAH] that can hydrate cyanamideto non-toxic urea.

pinII—indicates potato proteinase inhibitor. See Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498.

Pro—indicates a promoter sequence.

Term—indicates a terminator sequence.

Ubi Pro—indicates a ubiquitin promoter. See Christensen et al. (1989)Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol.Biol. 18:675-689) Ubi1ZM Pro-indicates a ubiquitin maize promoter.

Example 1

Seeds from haploid inducer lines, such as Stock 6, RWS, KEMS, KMS orZMS, are planted and self or sib pollinated at flowering. The ears aresurface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20minutes, and rinsed two times with sterile water.

For Agrobacterium-mediated transformation of maize the method of Zhao isemployed essentially as described in U.S. Pat. No. 5,981,840, thecontents of which are hereby incorporated by reference. Embryos(generally about 7-14 days after pollination) are isolated from a maizeplant and the embryos are contacted with a suspension of Agrobacterium,where the bacteria are capable of transferring the nucleotide sequencesof interest to at least one cell of at least one of the embryos. TheAgrobacterium comprises the following expression cassette.

Construct C:

ZM-lec1 promoter: UB1ZMintron:barstar:pinII 3′terminator—ZM-PG47promoter:ZM-BT1 transit peptide:ZM-alpha-amylase1:ZM-IN2-1 3′terminator—UB1ZM promoter:5′UTR intron:GAT:PINII 3′ terminator.

In this step the embryos are typically immersed in an Agrobacteriumsuspension for the initiation of inoculation. Preferably, theAgrobacterium suspension contains 100 μM acetosyringone. The embryos areco-cultured for a time with the Agrobacterium.

Generally the embryos are cultured on solid medium following theinfection step. Following this co-cultivation period an optional“resting” step lasting 6-7 days is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants. Next, inoculated embryos arecultured on solid medium containing a selective agent for GAT, which isthe herbicide glyphosate (GAT-genes encoding glyphosateN-acetyltransferase (GAT). See PCT publication WO02/36782 and U.S.application Ser. No. 10/427,692).

After being placed on the selection media the growing transformed callusis transformed with a second cassette, Construct D. This time usingparticle bombardment.

Construct D:

ZM-lec1 promoter:Potato LS intron:barnase:pinII 3′terminator UB1ZMpromoter:5′UTR intron:moPAT:PINII 3′ terminator.

This plasmid DNA is precipitated onto 1.1 μm (average diameter) tungstenpellets using a CaCl2 precipitation procedure as follows: 100 μlprepared tungsten particles in water, 10 μl (1 μg) DNA in TrisEDTAbuffer (1 μg total), 100 μl 2.5 M CaC1₂, 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 μl 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment. The sample plates are bombarded at level #4in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at650 PSI, with a total of ten aliquots taken from each tube of preparedparticles/DNA.

Following bombardment, the callus is kept on 560Y medium for 2 days,then transferred to selection medium containing the selection agent forthe PAT gene, bialophos, and subcultured every 2 weeks.

After approximately 10 weeks of selection, selection-resistant callusclones are transferred to regeneration medium to initiate plants.Following somatic embryo maturation (2-4 weeks), well-developed somaticembryos are transferred to medium for germination and transferred to thelighted culture room. Approximately 7-10 days later, developingplantlets are transferred to hormone-free medium in tubes for 7-10 daysuntil plantlets are well established. Plants are then transferred toinserts in flats (equivalent to 2.5″ pot) containing potting soil andgrown for 1 week in a growth chamber, subsequently grown an additional1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6gallon) and grown to maturity. Plants are monitored and scored for thegenotype and/or phenotype of interest.

Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl,120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought tovolume with D-I H20 following adjustment to pH 5.8 with KOH); 2.0 g/lGelrite (added after bringing to volume with D-I H20); and 8.5 mg/lsilver nitrate (added after sterilizing the medium and cooling to roomtemperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H20 following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H20); and 0.85 mg/lsilver nitrate and selection agent (both added after sterilizing themedium and cooling to room temperature).

Example 2 Production of Haploid Embryos

After an ablation haploid inducer line is produced as indicated inExample 1. The inducer line can be used to produce haploid embryos. Thiscan be achieved through the growing of F1 corn seed. For example a cornbreeder wanting to produce a superior inbred with the bestcharacteristics from elite inbred line A and elite inbred line B crossesthe two inbreds to form F1 hybrid seed. This F1 seed is grown along withthe ablation inducer line. The timing of the planting is such that thepollen of the ablation inducer line is ready at the silking time of theF1 hybrid seed. The silks of the F1 plants are pollinated with theablation inducer line pollen. This can be achieved by planting alternaterows of the F1 and the inducer line, and various combinations thereofwith the F1, or female, plants being detasseled before pollination. Thecrosses can also be done by shoot bagging and controlling pollination byhand pollinating.

After the seed has matured the seed can be harvested and the only viableseed will be the haploid seed. The haploid seed is then planted andundergoes chromosome doubling using a chromosomal doubling agent such aspronamide.

Example 3 Transformation and Regeneration of Transgenic Inducer MaizePlants

Hi-II maize seeds are planted. The ears at 9-12 days after pollinationare harvested surface sterilized in 30% Clorox bleach plus 0.5% Microdetergent for 20 minutes, and rinsed two times with sterile water. Theembryos are isolated from ears using a scalpel. Embryos are contactedwith a suspension of Agrobacterium, where the bacteria are capable oftransferring the nucleotide sequences of interest to at least one cellof at least one of the embryos. In this example the embryos areco-transformed with 2 Agrobacteria.

One Agrobacterium comprises the following expression cassette.

Construct E:

ZM-lec1 promoter: UB1ZMintron:barstar:pinII 3′terminator—ZM-PG47promoter:ZM-BT1 transit peptide:ZM-alpha-amylase1:ZM-IN2-1 3′terminator—UB1ZM promoter:5′UTR intron:GAT:PINII 3′ terminator.

The second Agrobacterium comprises the following expression cassette.

Construct F:

ZM-lec1 promoter:Potato LS intron:barnase:pinII 3′terminator—UB1ZMpromoter:5′UTR intron:moPAT:PINII 3′ terminator.

The barnase gene includes an intron for the purpose of preventingexpression of barnase in the Agrobacterium.

The tissue culture media and process is the same as described above withthe selection agent, bialophos. Callus that only comprises Construct Ewill be selected against with the use of bialophos. Callus that onlycomprises Construct F will not regenerate because the barstar constructis not available to prevent the toxicity of the barnase.

Plants will be regenerated that comprise Constructs E and F. Theseplants will be used to backcross both constructs into a maize haploidinducer line. During the backcross process the co-transformed lines willevaluated to determine the linkage between the constructs. The closerthe constructs are linked the less transmission of the barnasepolynucleotide through the pollen will occur because it will segregatewith the alpha-amylase polynucleotide expressed in the pollen making thepollen non-viable.

When a cross is made with unlinked constructs, the diploid embryos areeliminated because the barstar-containing construct is not transmittedthrough pollen, and they only inherit barnase.

Barstar and barnase are both expressed in early embryogenesis onseparate, unlinked transgenes in the haploid inducer line. Thus, thehaploid inducer line can be maintained.

Example 4 Transformation and Regeneration of Transgenic Inducer MaizePlants Using Agrobacterium Comprising Two T-DNA Constructs

One Agrobacterium comprises the following 2 expression cassettes.

RB:ZM-lec1 promoter: UB1ZMintron:barstar:pinII 3′terminator—ZM-PG47promoter:ZM-BT1 transit peptide:ZM-alpha-amylase1:ZM-IN2-1 3′terminator—UB1ZM promoter:5′UTR intron:GAT:PINII 3′ terminator:LBRB:ZM-lec1 promoter:Potato LS intron:barnase:pinII 3′terminator—UB1ZMpromoter:5′UTR intron:moPAT:PINII 3′ terminator:LBTransformation with Agrobacterium containing cassettes can increase theefficiency of producing stably transformed plants comprising the twocassettes at locations in the genome that are not linked (Miller et al.Transgenic Research 11:381-396, 2002. High efficiency transgenesegregation in co-transformed maize plants using an Agrobacteriumtumefaciens 2 T-DNA binary system.)

Other than the vector, the process to obtain a maize ablation haploidinducer line can be the same as indicated in Example 3.

1. (canceled)
 2. A method of selecting for haploid embryos comprising:a) Pollinating a first maize plant with the pollen from a maize haploidinducer line to produce embryos wherein said maize haploid inducer linecomprises an embryo expressed lethal polynucleotide, a non-transmissionpollen polynucleotide, and an embryo expressed inhibitor polynucleotidethat prevents the lethality of said embryo expressed lethalpolynucleotide; b) producing haploid embryos and non-viable diploidembryos; and c) selecting haploid maize embryos; further wherein saidembryo expressed lethal polynucleotide is barnase and said embryoexpressed inhibitor polynucleotide is barstar.
 3. (canceled) 4.(canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. A transgenicmaize haploid inducer line comprising a first and second expressioncassette wherein said first expression cassette comprises apolynucleotide that is lethal to embryos and wherein said secondcassette is not transmitted through pollen and comprises apolynucleotide that inhibits the lethality of said polynucleotide thatis lethal to embryos, wherein said polynucleotide that is lethal toembryos is a barnase polynucleotide and said polynucleotide thatinhibits the lethality of said polynucleotide that is lethal to embryosis a barstar polynucleotide.
 9. The transgenic maize haploid inducerline of claim 8 wherein said polynucleotide that is lethal to embryoscomprises a lec1 promoter.
 10. (canceled)
 11. (canceled)
 12. (canceled)13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled) 17.(canceled)
 18. (canceled)
 19. A transgenic maize haploid inducer linecomprising a first and second expression cassette wherein; said firstexpression cassette comprises a barnase polynucleotide that is operablylinked to a lec1 promoter and is lethal to embryos and wherein; saidsecond cassette comprises a barstar polynucleotide that inhibits thelethality of said polynucleotide that is lethal to embryos and wherein,said second expression cassette also comprises an alpha-amylasepolynucleotide operably linked to a PG47 promoter wherein expression ofsaid alpha-amylase polynucleotide operably linked to a PG47 promoterdoes not allow pollen transmission.